Presence of SARS-Coronavirus-2 in sewage

Gertjan Medema; Leo Heijnen; Goffe Elsinga; Ronald Italiaander; Anke Brouwer

The recovery of F-specific RNA phages by the purification and concentration steps was 73  50% (n=16). Initial trials showed inhibition of the RT-PCR reactions, that could be reduced to a large extend by the addition of BSA to the reaction mixture. The results of the samples of February 6, 2020, three weeks before the first case was reported in The Netherlands on Feb 27, showed no positive signals for primer sets N1-3 and E ( Table 3 ). The samples of March 4 and 5, one week into the epidemic with 38 and 82 COVID-19 cases reported through the health surveillance system, respectively 19 , showed a positive signal for the N1 primer/probe set in sewage samples of four of the 6 WWTP sampled. In sewage samples of March 15 and 16, 6 of the 7 WWTP showed a positive signal with N1, and in addition 5 WWTP also with N3 and 4 WWTP with the E primer/probe set. High throughput electrophoresis confirmed that the length of the PCR products match the length of the PCR target gene fragments. N1 appears to be the most sensitive primer/probe set of the sets tested here, followed by N3 and E for detection of SARS-CoV-2 in sewage. For clinical samples, the US FDA reported the sensitivity of the primer/probe sets of N1=N3>N2 on SARS-CoV-2 RNA, 20 which partially matches with our results in sewage samples. The detection of fragments of two genes of SARS-CoV-2 in sewage of multiple WWTP and the temporal pattern of detection that aligns with the emergence of the epidemic in the Netherlands provide compelling evidence that SARS-CoV-2 is detected in sewage.